Mitochondrial DNA in yeast and some mammalian species.

Yeast DNA, in a cesium chloride density gradient, shows a minor or satellite band with a density lower than that of the main nuclear component. The DNA isolated from purified mitochondria of yeasts corresponds in density to this satellite band. In solution, this DNA more easily undergoes renaturation as compared to DNA from cell nuclei. The ease of this renaturation is presumably due to a homogeneity greater than that of nuclear DNA. Mitochondrial DNA isolated from several mammalian species has the same or higher density than nuclear DNA, but differs in its ready renaturability.

Energy-linked and energy-independent transhydrogenase activities in Escherichia coli vesicles.

Active transport vesicles of Escherichia coli were shown to possess low levels of energy-independent and energy-dependent nicotinamide nucleotide transhydrogenase activities. Breakage of such vesicles in a French pressure cell resulted in a fraction which had an 8-10-fold increased respiration- and ATP-driven transhydrogenase activities. Stimulation of the ATPase activity in vesicles with Triton X-100 was also paralledled by a 2-fold increase in the energy-independent transhydrogenase. Disruption of the vesicles similarly resulted in increases in the energy-independent transhydrogenase, NADH and succinate oxidase activities but a decrease in succinate supported proline uptake. In the light of these findings, the "sidedness' of the vesicle membranes is discussed.

Occurrence of proteins immunoreactive with anti-coupling factor B in phosphorylating membrane preparations.

Coupling factor B has been isolated from beef heart mitochondria, apparently in multiple forms which differ in molecular weight and specific activity. Since it has no known intrinsic catalytic activity, detection and quantitation have been based upon the factor B-dependent stimulation of ATP-linked activities in factor B-deficient sub-mitochondrial particles. This communication reports the development of a reliable and more universally applicable enzyme-linked immunosorbent assay (ELISA) for detection and quantitation of factor B in soluble or membranous preparations. The assay requires nanoliter volumes of rabbit antiserum raised against purified factor B and will detect nanogram amounts of the coupling factor. Analysis of beef heart submitochondrial particles using a competitive binding ELISA indicated a factor B content of 0.27 nmol/mg protein, making factor B stoichiometric with F1 (0.3--0.6 nmol/mg). Furthermore, application of the factor B ELISA has indicated the presence of material cross-reacting with the beef heart factor B-antiserum in phosphorylating membranes from chloroplasts, Escherichia coli, Paracoccus denitrificans and the thermophilic bacterium, PS3. Negative results were obtained with mitochondria and microsomes from rat liver, purple membranes from Halobium halobacterium and sarcoplasmic reticulum from rabbit skeletal muscle.

Human mitochondrial ATP synthase: cloning cDNA for the nuclear-encoded precursor of coupling factor 6.

Coupling factor 6 (F6) is a component of mitochondrial ATP synthase which is required for the interactions of the catalytic and proton-translocating segments. A human fetal muscle cDNA clone encoding this protein was isolated by screening a lambda gt10 library with oligodeoxyribonucleotide probes. The 497-bp F6 cDNA included a 96-bp segment that delineated a presequence of 32 amino acids (aa) in the precursor protein, and 140 bp of 3'-untranslated sequence. The remainder of the cDNA sequence coded for a mature human F6 protein of 76 aa. The deduced primary aa sequence showed 81% homology to that of bovine F6, differing in 14 aa. Almost all of these aa substitutions were conservative and comparison of the hydropathy profiles revealed a similar pattern.

Diamide blocks H+ conductance in mitochondrial H+-ATPase by oxidizing FB dithiol.

Effects of diamide on proton conductance of electron transport particles (ETPH), purified H+-ATPase (F1-F0), F0 of the H+-ATPase from beef heart mitochondria and binding of cadmium (109Cd) to the H+-ATPase have been examined in the present paper. When ETPH and purified H+-ATPase are treated with 1 mM diamide, ATP-dependent generation of membrane potential, monitored by the absorbance change produced by the redistribution of oxonol VI, is consistently inhibited. Diamide also blocks passive H+ conductance driven by a K+ diffusion potential in the membrane sector, F0, of H+-ATPase. Furthermore, diamide treatment drastically reduces the binding of 109Cd2+ to H+-ATPase, showing competition for the FB dithiol group.

Monoclonal antibodies to mitochondrial coupling factor B.

Two monoclonal antibodies (MAb I and IV) have been prepared which showed high and specific reactions towards bovine heart mitochondrial coupling factor B (FB). Both have been identified as sub-type IgG1 of mouse immunoglobulins. MAb I reacts with purified and functionally active FB, alkylated or oxidized forms of FB and even with peptides formed on digestion of FB with trypsin. When used together, MAb I and IV reacted with FB in immunoblots of normal and urea treated samples of mitochondria, submitochondrial particles, ammonia-EDTA extracted particles, and H+-ATPase. Both MAbs inhibited FB-stimulated ATP-dependent reverse electron flow activity when FB was incubated with the antibody either before or after its addition to FB-deficient AE-particles. Reactivity of MAb I towards FB declined upon exposure of FB to guanidine HC1 while reactivity of MAb IV remained unaltered.

Amino-terminal amino acid sequence of beef heart mitochondrial coupling factor B.

Bovine heart mitochondrial coupling factor B was isolated and purified to homogeneity in its active form. The amino-terminal amino acid sequence of the alkylated protein was determined. Two chains with exactly the same sequence except for the presence of an additional Phe at the amino-terminus on one of them were obtained. The 55 amino acid sequence appears to be largely hydrophilic with several charged amino acid residues. This sequence showed no homology with the E. coli unc operon, oligomycin sensitivity conferring protein, or coupling factor 6 or any protein in the data base.

A simple method for maintaining large, aging populations of Caenorhabditis elegans.

This paper describes a technique capable of establishing and maintaining large, age-synchronous populations of the nematode Caenorhabditis elegans. The technique has three essential components: a rich chemical medium; a method for producing and harvesting mass quantities of eggs; and 5-fluorodeoxyuridine (FUdR), an inhibitor of DNA synthesis. A culture of worms is filtered through glass wool or a wire screen to isolate young larvae. Eggs laid by these worms after they mature are collected over a period of 4-6 hours and allowed to hatch. A low level of FUdR (25 microM) is added just before the larvae reach maturity. This timing is important to avoid developmental abnormalities. The adults lay eggs in the presence of FUdR but the eggs do not hatch, which maintains the synchrony of the culture. Many aging characteristics appear to be similar in treated and untreated worms, such as the time of cessation of egg production, the appearance of visible and behavioral age-related changes, and the mean lifespan. This system thus seems suitable for large-scale biochemical analysis of certain aspects of aging in C. elegans.

Inhibition by dicyclohexylcarbodiimide of ATP synthesis in isolated rat hepatocytes.

Dicyclohexylcarbodiimide (DCCD) inhibits, by 50%, ATP synthesis in isolated hepatocytes. This inhibition is associated with DCCD-binding to a proteolipid fraction present in submitochondrial particles.